MaxOneを用いた脳スライス実験 
MaxOneは研究者に生体外インタクト脳ネットワークをラベルフリーで分析することを可能にします。
- 急性脳スライス
- 器官型脳スライス培養とオルガノイド
- 生体外脳サンプルの作成 (例 亀の眼球ー脳のサンプル)
プロトコル
記録のための急性脳スライスサンプルの作成
- 解剖そしてスライス後、脳スライスを人工脳脊髄液(ACSF)内で、30-45分間、35度に保ち培養します。その後、記録するまでスライスを室温に保ちます。
- MaxOneアレイ上に急性スライスを置き、ピペットで可能な限り液体を吸い取ります。組織ホルダーでアレイ上の組織を軽く押し平らにします。膜が組織に接触しているか確認してください。
- 急性スライスをカルボゲンを充填した人工脳脊髄液(31-33度)を灌流させます。実験中、人工脳脊髄液の灌流を続けます。
- 解剖後4-6時間以内に、高密度微小電極アレイで記録および刺激実験を行います。
人工脳脊髄液の含有物(単位mM):
塩化ナトリウム 125、塩化カリウム 2.5、リン酸二水素ナトリウム 1.25、硫酸マグネシウム 1.9、グルコース 20、炭酸水素ナトリウム 25
このようなことができます
単一神経細胞およびネットワーク全体のフィールド電位の捕獲
MEA上の活性神経細胞から高品質信号の記録
MaxOneは神経細胞活動を高い時空間解像度で多重スケールで記録することが可能です。
- 局所フィード電位とスパイクの両方をインタクトブレインネットワークから同時に検出することができます。
- 低ノイズ信号は実験から浮かび上がる神経活動の特徴の抽出を促進します。
- 脳全体のフィールド電位伝播を捕らえ解析が可能です。
細胞とシナプスを介したつながりをラージスケールマッピング
脳組織内のすべての活性神経細胞の活動電位空間場、軸索突起およびシナプス後の信号を抽出、解析します。MaxOneは脳スライス内のスパイクしている神経細胞を検出し、電気的な刺激によって細胞神経活動を誘発することが可能です。
- 脳スライス上でスパイクするニューロンを検知することで神経活動マップを抽出できます。
- スパイク頻度
- シナプス後の事象はスパイク後の緩やかな +/- シグナルで表したスパイク誘発の平均化によって明らかになります。(M. Shein-Idelson 他, Nature Methods, 2017)
MaxOne 組織ホルダー
MaxOne組織ホルダーはMEA上の脳スライスを平坦にすることで、安定した再現性のある実験を可能にしますに。細胞ホルダーは灌流溶液下で実験中、MEA上に組織を押して平らにし固定し続けることができます。
詳細はこちら論文–
脳スライスの応用分野
Al-Absi, Abdel-Rahman; Thambiappaa, Sakeerthi Kethees; Khanc, Ahmad Raza; Glerup, Simon; Sanchez, Connie; Landau, Anne M; Nyengaard, Jens R Molecular and Cellular Neuroscience, 2022. Kajiwara Motoki; Nomura, Ritsuki; Goetze Felix; Kawabata Masanori; Isomura Yoshikazu; Akutsu Tatsuya; Shimono Masanori; Inhibitory neurons exhibit high controlling ability in the cortical microconnectome Journal Article PLOS Computational Biology, 2021. Obien, Marie Engelene J; Hierlemann, Andreas; Frey, Urs Accurate signal-source localization in brain slices by means of high-density microelectrode arrays Journal Article Scientific Reports, 9 (788), 2019. Shein-Idelson, Mark; Pammer, Lorenz; Hemberger, Mike; Laurent, Gilles Large-scale mapping of cortical synaptic projections with extracellular electrode arrays Journal Article Nature Methods, 14 (9), pp. 882–889, 2017, ISSN: 1548-7091. Viswam, Vijay; Bounik, Raziyeh; Shadmani, Amir; Dragas, Jelena; Obien, Marie Engelene J; Muller, Jan; Chen, Yihui; Hierlemann, Andreas 19th International Conference on Solid-State Sensors, Actuators and Microsystems (TRANSDUCERS) Kaohsiung, Taiwan, 2017, ISSN: 2167-0021. Gong, Wei; Sencar, Jure; Bakkum, Douglas J; Jäckel, David; Obien, Marie Engelene J; Radivojevic, Milos; Hierlemann, Andreas Multiple single-unit long-term tracking on organotypic hippocampal slices using high-density microelectrode arrays Journal Article Frontiers in Neuroscience, 10 , pp. 1-16, 2016, ISSN: 1662453X. Frey, Urs; Egert, Ulrich; Heer, Flavio; Hafizovic, Sadik; Hierlemann, Andreas Microelectronic system for high-resolution mapping of extracellular electric fields applied to brain slices Journal Article Biosensors and Bioelectronics, 24 (7), pp. 2191-2198, 2009, ISSN: 09565663.
title = {Df(h22q11)/+ mouse model exhibits reduced binding levels of GABAA receptors and structural and functional dysregulation in the inhibitory and excitatory networks of hippocampus},
author = {Abdel-Rahman Al-Absi and Sakeerthi Kethees Thambiappaa and Ahmad Raza Khanc and Simon Glerup and Connie Sanchez and Anne M. Landau and Jens R. Nyengaard},
url = {https://www.sciencedirect.com/science/article/pii/S1044743122000756?via%3Dihub},
doi = {https://doi.org/10.1016/j.mcn.2022.103769},
year = {2022},
date = {2022-08-18},
journal = {Molecular and Cellular Neuroscience},
abstract = {The 22q11.2 hemizygous deletion confers high risk for multiple neurodevelopmental disorders. Inhibitory signaling, largely regulated through GABAA receptors, is suggested to serve a multitude of brain functions that are disrupted in the 22q11.2 deletion syndrome.
We investigated the putative deficit of GABAA receptors and the potential substrates contributing to the inhibitory and excitatory dysregulations in hippocampal networks of the Df(h22q11)/+ mouse model of the 22q11.2 hemizygous deletion. The Df(h22q11)/+ mice exhibited impairments in several hippocampus-related functional domains, represented by impaired spatial memory and sensory gating functions. Autoradiography using the [3H]muscimol tracer revealed a significant reduction in GABAA receptor binding in the CA1 and CA3 subregions, together with a loss of GAD67+ interneurons in CA1 of Df(h22q11)/+ mice. Furthermore, electro- physiology recordings exhibited significantly higher neuronal activity in CA3, in response to the GABAA receptor antagonist, bicuculline, as compared with wild type mice. Density and volume of dendritic spines in pyramidal neurons were reduced and Sholl analysis also showed a reduction in the complexity of basal dendritic tree in CA1 and CA3 subregions of Df(h22q11)/+ mice.
Overall, our findings demonstrate that hemizygous deletion in the 22q11.2 locus leads to dysregulations in the inhibitory circuits, involving reduced binding levels of GABAA receptors, in addition to functional and structural modulations of the excitatory networks of hippocampus.},
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We investigated the putative deficit of GABAA receptors and the potential substrates contributing to the inhibitory and excitatory dysregulations in hippocampal networks of the Df(h22q11)/+ mouse model of the 22q11.2 hemizygous deletion. The Df(h22q11)/+ mice exhibited impairments in several hippocampus-related functional domains, represented by impaired spatial memory and sensory gating functions. Autoradiography using the [3H]muscimol tracer revealed a significant reduction in GABAA receptor binding in the CA1 and CA3 subregions, together with a loss of GAD67+ interneurons in CA1 of Df(h22q11)/+ mice. Furthermore, electro- physiology recordings exhibited significantly higher neuronal activity in CA3, in response to the GABAA receptor antagonist, bicuculline, as compared with wild type mice. Density and volume of dendritic spines in pyramidal neurons were reduced and Sholl analysis also showed a reduction in the complexity of basal dendritic tree in CA1 and CA3 subregions of Df(h22q11)/+ mice.
Overall, our findings demonstrate that hemizygous deletion in the 22q11.2 locus leads to dysregulations in the inhibitory circuits, involving reduced binding levels of GABAA receptors, in addition to functional and structural modulations of the excitatory networks of hippocampus.
title = {Inhibitory neurons exhibit high controlling ability in the cortical microconnectome},
author = {Kajiwara, Motoki; Nomura, Ritsuki; Goetze, Felix; Kawabata, Masanori; Isomura, Yoshikazu; Akutsu, Tatsuya; Shimono, Masanori; },
url = {https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1008846},
year = {2021},
date = {2021-04-08},
journal = {PLOS Computational Biology},
abstract = {The brain is a network system in which excitatory and inhibitory neurons keep activity bal- anced in the highly non-random connectivity pattern of the microconnectome. It is well known that the relative percentage of inhibitory neurons is much smaller than excitatory neu- rons in the cortex. So, in general, how inhibitory neurons can keep the balance with the sur- rounding excitatory neurons is an important question. There is much accumulated knowledge about this fundamental question. This study quantitatively evaluated the rela- tively higher functional contribution of inhibitory neurons in terms of not only properties of individual neurons, such as firing rate, but also in terms of topological mechanisms and con- trolling ability on other excitatory neurons. We combined simultaneous electrical recording (~2.5 hours) of ~1000 neurons in vitro, and quantitative evaluation of neuronal interactions including excitatory-inhibitory categorization. This study accurately defined recording brain anatomical targets, such as brain regions and cortical layers, by inter-referring MRI and immunostaining recordings. The interaction networks enabled us to quantify topological influence of individual neurons, in terms of controlling ability to other neurons. Especially, the result indicated that highly influential inhibitory neurons show higher controlling ability of other neurons than excitatory neurons, and are relatively often distributed in deeper layers of the cortex. Furthermore, the neurons having high controlling ability are more effectively limited in number than central nodes of k-cores, and these neurons also participate in more clustered motifs. In summary, this study suggested that the high controlling ability of inhibi- tory neurons is a key mechanism to keep balance with a large number of other excitatory neurons beyond simple higher firing rate. Application of the selection method of limited important neurons would be also applicable for the ability to effectively and selectively stimu- late E/I imbalanced disease states.},
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title = {Accurate signal-source localization in brain slices by means of high-density microelectrode arrays},
author = {Marie Engelene J. Obien and Andreas Hierlemann and Urs Frey},
url = {https://www.nature.com/articles/s41598-018-36895-y},
doi = {10.1038/s41598-018-36895-y},
year = {2019},
date = {2019-01-28},
journal = {Scientific Reports},
volume = {9},
number = {788},
abstract = {Extracellular recordings by means of high-density microelectrode arrays (HD-MEAs) have become a powerful tool to resolve subcellular details of single neurons in active networks grown from dissociated cells. To extend the application of this technology to slice preparations, we developed models describing how extracellular signals, produced by neuronal cells in slices, are detected by microelectrode arrays. The models help to analyze and understand the electrical-potential landscape in an in vitro HD-MEA-recording scenario based on point-current sources. We employed two modeling schemes, (i) a simple analytical approach, based on the method of images (MoI), and (ii) an approach, based on finite-element methods (FEM). We compared and validated the models with large-scale, high-spatiotemporal-resolution recordings of slice preparations by means of HD-MEAs. We then developed a model-based localization algorithm and compared the performance of MoI and FEM models. Both models provided accurate localization results and a comparable and negligible systematic error, when the point source was in saline, a condition similar to cell-culture experiments. Moreover, the relative random error in the x-y-z-localization amounted only up to 4.3% for z-distances up to 200 μm from the HD-MEA surface. In tissue, the systematic errors of both, MoI and FEM models were significantly higher, and a pre-calibration was required. Nevertheless, the FEM values proved to be closer to the tissue experimental results, yielding 5.2 μm systematic mean error, compared to 22.0 μm obtained with MoI. These results suggest that the medium volume or “saline height”, the brain slice thickness and anisotropy, and the location of the reference electrode, which were included in the FEM model, considerably affect the extracellular signal and localization performance, when the signal source is at larger distance to the array. After pre-calibration, the relative random error of the z-localization in tissue was only 3% for z-distances up to 200 μm. We then applied the model and related detailed understanding of extracellular recordings to achieve an electrically-guided navigation of a stimulating micropipette, solely based on the measured HD-MEA signals, and managed to target spontaneously active neurons in an acute brain slice for electroporation.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
title = {Large-scale mapping of cortical synaptic projections with extracellular electrode arrays},
author = {Mark Shein-Idelson and Lorenz Pammer and Mike Hemberger and Gilles Laurent},
url = {http://www.nature.com/doifinder/10.1038/nmeth.4393},
doi = {10.1038/nmeth.4393},
issn = {1548-7091},
year = {2017},
date = {2017-08-14},
journal = {Nature Methods},
volume = {14},
number = {9},
pages = {882--889},
abstract = {Understanding circuit computation in the nervous system requires sampling activity over large neural populations and maximizing the number of features that can be extracted. By combining planar arrays of extracellular electrodes with the three-layered cortex of turtles, we show that synaptic signals induced along individual axons as well as action potentials can be easily captured. Two types of information can be extracted from these signals, the neuronal subtype (inhibitory or excitatory)—whose identification is more reliable than with traditional measures such as action potential width—and a (partial) spatial map of functional axonal projections from individual neurons. Because our approach is algorithmic, it can be carried out in parallel on hundreds of simultaneously recorded neurons. Combining our approach with soma triangulation, we reveal an axonal projection bias among a population of pyramidal neurons in turtle cortex and confirm this bias through anatomical reconstructions.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
title = {High-density Mapping of Brain Slices Using a Large Multi-functional High-density CMOS Microelectrode Array System},
author = {Vijay Viswam and Raziyeh Bounik and Amir Shadmani and Jelena Dragas and Marie Engelene J. Obien and Jan Muller and Yihui Chen and Andreas Hierlemann },
url = {https://ieeexplore.ieee.org/abstract/document/7994006},
doi = {10.1109/TRANSDUCERS.2017.7994006},
issn = {2167-0021},
year = {2017},
date = {2017-06-18},
pages = {135-138},
address = {Kaohsiung, Taiwan},
organization = {19th International Conference on Solid-State Sensors, Actuators and Microsystems (TRANSDUCERS)},
abstract = {We present a CMOS-based high-density microelectrode array (HD-MEA) system that enables high-density mapping of brain slices in-vitro with multiple readout modalities. The 4.48×2.43 mm 2 array consists of 59,760 micro-electrodes at 13.5 μm pitch (5487 electrodes/mm 2 ). The overall system features 2048 action-potential, 32 local-field-potential and 32 current recording channels, 32 impedance-measurement and 28 neurotransmitter-detection channels and 16 voltage/current stimulation channels. The system enables real-time and label-free monitoring of position, size, morphology and electrical activity of brain slices.},
keywords = {},
pubstate = {published},
tppubtype = {conference}
}
title = {Multiple single-unit long-term tracking on organotypic hippocampal slices using high-density microelectrode arrays},
author = {Wei Gong and Jure Sencar and Douglas J Bakkum and David Jäckel and Marie Engelene J Obien and Milos Radivojevic and Andreas Hierlemann},
url = {https://www.frontiersin.org/articles/10.3389/fnins.2016.00537/full},
doi = {10.3389/fnins.2016.00537},
issn = {1662453X},
year = {2016},
date = {2016-11-22},
journal = {Frontiers in Neuroscience},
volume = {10},
pages = {1-16},
abstract = {A novel system to cultivate and record from organotypic brain slices directly on high-density microelectrode arrays (HD-MEA) was developed. This system allows for continuous recording of electrical activity of specific individual neurons at high spatial resolution while monitoring at the same time, neuronal network activity. For the first time, the electrical activity patterns of single neurons and the corresponding neuronal network in an organotypic hippocampal slice culture were studied during several consecutive weeks at daily intervals. An unsupervised iterative spike-sorting algorithm, based on PCA and k-means clustering, was developed to assign the activities to the single units. Spike-triggered average extracellular waveforms of an action potential recorded across neighboring electrodes, termed ‘footprints' of single-units were generated and tracked over weeks. The developed system offers the potential to study chronic impacts of drugs or genetic modifications on individual neurons in slice preparations over extended times.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
title = {Microelectronic system for high-resolution mapping of extracellular electric fields applied to brain slices},
author = {Urs Frey and Ulrich Egert and Flavio Heer and Sadik Hafizovic and Andreas Hierlemann},
url = {http://www.sciencedirect.com/science/article/pii/S095656630800643X?via%3Dihub},
doi = {10.1016/j.bios.2008.11.028},
issn = {09565663},
year = {2009},
date = {2009-03-15},
journal = {Biosensors and Bioelectronics},
volume = {24},
number = {7},
pages = {2191-2198},
abstract = {There is an enduring quest for technologies that provide - temporally and spatially - highly resolved information on electric neuronal or cardiac activity in functional tissues or cell cultures. Here, we present a planar high-density, low-noise microelectrode system realized in microelectronics technology that features 11,011 microelectrodes (3,150 electrodes per mm2), 126 of which can be arbitrarily selected and can, via a reconfigurable routing scheme, be connected to on-chip recording and stimulation circuits. This device enables long-term extracellular electrical-activity recordings at subcellular spatial resolution and microsecond temporal resolution to capture the entire dynamics of the cellular electrical signals. To illustrate the device performance, extracellular potentials of Purkinje cells (PCs) in acute slices of the cerebellum have been analyzed. A detailed and comprehensive picture of the distribution and dynamics of action potentials (APs) in the somatic and dendritic regions of a single cell was obtained from the recordings by applying spike sorting and spike-triggered averaging methods to the collected data. An analysis of the measured local current densities revealed a reproducible sink/source pattern within a single cell during an AP. The experimental data substantiated compartmental models and can be used to extend those models to better understand extracellular single-cell potential patterns and their contributions to the population activity. The presented devices can be conveniently applied to a broad variety of biological preparations, i.e., neural or cardiac tissues, slices, or cell cultures can be grown or placed directly atop of the chips for fundamental mechanistic or pharmacological studies.},
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pubstate = {published},
tppubtype = {article}
}